1. Field of the Invention
The present invention relates to processes for the production of podophyllotoxin compounds by culturing Podophyllum, and to the preparation of an adventive embryo useful for such processes.
2. Description of the Related Art
Perennial plants belonging to the genus Podophyllum are important foliage plants, and recently have been found to produce podophyllotoxin compounds and flavonoid compounds which are useful in the fields of pharmaceuticals and cosmetics, and therefore, have become industrially valuable. For example, podophyllotoxin, one of the podophyllotoxin compounds, was found to have an aperitive activity and antimicrobial activity, and has been used as an aperitive medicament or antimicrobial agent. Moreover, recently, podophyllotoxin has attracted attention as an anti-tumor agent, and the development of anti-tumor agents derived from podophyllotoxin compounds is underway.
Nevertheless, in the industrial production of the podophyllotoxin compounds many problems arise, as in the industrial production of other substances of a higher plant origin. Namely, although to produce such substances it is necessary to obtain a naturally occurring desired plant and to-cultivate the plant, the production of the desired product by cultivating the plant is accompanied by various limitations. For example, the productivity of a desired product varies according to various conditions, including environmental conditions, and in general the productivity is relatively low. Therefore, it is very difficult to consistently obtain a large amount of Podophyllum plant as an industrial starting material for the production of podophyllotoxin compounds.
To resolve the above-mentioned problems accompanying the use of the plant, the use of a plant tissue culture has been proposed. In the tissue culture method, cells or tissue such as callus, dedifferentiated cells, adventive embryo, adventitious organ, or the like are cultured in a medium to accumulate a desired product in the culture medium, which product is then recovered.
General procedures for generating and culturing the above-mentioned plant entities are known, but particular conditions for the generation and culturing of the plant entities differ depending on the plant, and conditions suitable for one particular plant are not necessarily suitable for another plant. Further, the conditions for the production of a particular substance are very strict, and conditions suitable for a particular producer plant, or for a particular product, cannot be automatically applied to the production of an other product or an other plant. Therefore, the conditions for the production must be experimentally determined for each particular plant and each particular product.
Japanese Unexamined Patent Publication No. 62-96088 describes a process for obtaining podophyllotoxin compounds in an amount of 0.0035% by weight relative to the plant mass by culturing an explant of a Podophyllum plant on a solid medium to form a callus, which is then cultured on a solid medium to form a large amount of callus/adventitious roots, and extracting podophyllotoxin compounds from the roots. According to this method, however, the preparation of a large amount of adventitious roots is not always easy, and an accumulated amount of podophyllotoxin compounds is not always satisfactory.
An adventive embryo is useful as a starting or intermediate material for a plant tissue culture for the production of a desired substance, although it is also industrially useful as a starting or intermediate material for the mass-proliferation of plants, in the interests of an efficient breeding and efficient cultivation of the plants, and as a main component of artificial seeds.
Nevertheless, it is very difficult to reproducibly generate an adventive embryo, and to date, the generation of an adventive embryo has been reported only for a very limited plant species. Moreover, in most cases, since the generation of an adventive embryo is carried out on a solid medium, the generation of the adventive embryo takes a relatively long time and the recovery of the adventive embryo is difficult, resulting in an impracticality for an industrial application.
To obtain an adventive embryo as a starting material of the proliferation and tissue culture of useful plants, various methods have been proposed. According to one method, non-differentiated cells of a desired plant are cultured in the presence of plant growth hormones such as auxin to induce the generation of an adventive embryo. According to another method, an osmotic pressure of a medium for culturing a non-differentiated cell is changed to induce the generation of an adventive embryo without using plant growth modulators. These methods however, can not be universally applied to all plants, since the conditions for the generation of an adventive embryo differ depending on particular plants. Further, the generation of an adventive embryo of plants belonging to the genus Podophyllum is not known.